This invention related generally to vitamin D compounds and in particular, to vitamin D compounds in which the C-25 or equivalent position is double bonded, and specifically to a method of making such compounds.
Vitamin D has long been established as having an important biological role in bone and mineral metabolism. For example, vitamin D plays a critical role in stimulating calcium absorption and regulating calcium metabolism. It is also known that it is not vitamin D itself, but metabolites generated from it in vivo that are effective in regulating calcium metabolism. The discovery of active forms of vitamin D, (M. F. Holick et al., 68 Proc. Natl. Acad. Sci. USA, 803-804 (1971); G. Jones et al., 14 Biochemistry, 1250-1256 (1975), and other active vitamin D analogs (M. F. Holick et al., 180 Science 190-191 (1973); H. Y. Lam et al., 186 Science 1038-1040 (1974) caused much excitement and speculation about the usefulness of these vitamin D compounds in the treatment of bone depletive disorders.
Animal studies examining the effects of these active vitamin D compounds, particularly 1xcex1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D3, suggested that such agents would be useful in restoring calcium balance. An early clinical study indicated that oral administration of 0.5 xcexcg/day of 1xcex1,25-dihydroxyvitamin D3 to a group of postmenopausal women improved intestinal calcium absorption as well as calcium balance in the women. On this basis, U.S. Pat. No. 4,225,596 (xe2x80x9c""596 Patentxe2x80x9d) described and claimed the use of 1xcex1,25-dihydroxyvitamin D3 for increasing calcium absorption and retention, i.e., this compound is highly potent in stimulating intestinal calcium absorption as well as the resorption of calcium from bone (i.e., bone mobilization).
The best indicator of the efficacy of vitamin D compounds, however, in the prevention or treatment of depletive bone disorders, however, is bone itself rather than calcium absorption or calcium balance. More recent clinical data indicate that, at the dosage ranges taught in the ""596 Patent, 1xcex1,25-dihydroxy-vitamin D3 has, at best, modest efficacy in preventing or restoring loss of bone mass or bone mineral content (S. M. Ott and C. H. Chesnut, 110 Ann. Int. Med. 267-274 (1989); J. C. Gallagher et al., 113 Ann. Int. Med. 649-655 (1990); J. Aloia et al., 84 Amer. J. Med. 401-408 (1988)).
These clinical studies with 1xcex1,25-dihydroxyvitamin D3, and another conducted with 1xcex1-hydroxyvitamin D3 (M. Shiraki et al., 32 Endocrinol. Japan 305-315 (1985)), indicate that the capacity of these two vitamin D compounds to restore lost bone mass or bone mineral content is dose-related. The studies also indicate, however, that, at the dosage ranges required for either compound to be truly effective, toxicity in the form of hyperclacemia and hypercalciuria becomes a major problem. Specifically, attempts to increase the amount of 1xcex1,25-dihydroxyvitamin D3 above 0.5 xcexcg/day have frequently resulted in toxicity. At dosage levels below 0.5 xcexcg/day, no effects are observed on bone mass or mineral content. (See, G. F. Jensen et al., 16 Clin. Invest. 305-309 (1981)). Two xcexcg/day of 1xcex1-hydroxyvitamin D3 was found to have efficacy in increasing bone mass in patients exhibiting senile osteoporosis (O. H. Sorensen et al., 7 Clin. Endocrinol. 169S-175S (1977)). Data from clinical studies in Japan, a population that has low calcium intake, indicate that efficacy is found with 1xcex1-hydroxyvitamin D3 when administered at 1 xcexcg/day (M. Shiraki et al., 32 Endocrinol. Japan. 305-315 (1985); H. Orimo et al., 3 Bone and Mineral 47-52 (1987)). At 2 xcexcg/day, however, toxicity with 1xcex1-hydroxyvitamin D3 occurs in approximately 67 percent of the patients, and at 1 xcexcg/day, this percentage is approximately 20 percent. Thus, the 1xcex1-hydroxylated vitamin D3 compounds can produce dangerously elevated blood calcium levels due to their inherent calcemic activity.
Because of their toxicity, 1-hydroxylated vitamin D3 compounds can only be administered in oral dosages that are, at best, modestly beneficial in preventing or treating loss of bone or bone mineral content. Indeed, Aloia recommends that alternative routes of administration be sought which might avoid the toxicity problems and allow higher dosage levels to be achieved. (J. Aloia et al., 84 Amer. J. Med. 401-408 (1988)). Despite reported toxicities of 1xcex1-hydroxyvitamin D3 and 1xcex1,25-dihydroxyvitamin D3, these two compounds remain the drugs of choice for many bone depletive disease treatments and calcium metabolism disorders such as renal osteodystrophy, hypoparathyroidism, vitamin D-resistant rickets and osteoporosis.
These two drugs also remain the only approved forms of 1xcex1-hydroxylated vitamin D for treating or preventing hyperparathyroidism which occurs secondary to the renal disease, although both drugs are not currently approved in all major pharmaceutical markets.
More recently, in addition to vitamin D""s role in regulating calcium homeostatis, other biological roles for vitamin D have come to light. Specific nuclear receptors for 1xcex1,25-dihydroxyvitamin D3 have been found in cells from diverse organs not involved in calcium homeostasis. For example, Miller et al., 52 Cancer Res. (1992) 515-520, have demonstrated biologically active, specific receptors for 1,xcex125-dihydroxyvitamin D3 in the human prostatic carcinoma cell line, LNCaP.
It has been also shown that certain vitamin D compounds and analogs are potent inhibitors of malignant cell proliferation and inducers/stimulators of cell differentiation. For example, U.S. Pat. No. 4,391,802 issued to Suda et al. discloses that 1xcex1-hydroxyvitamin D compounds, specifically 1xcex1,25-dihydroxyvitamin D3 and 1xcex1-hydroxyvitamin D3, possess potent antileukemic activity by virtue of inducing the differentiation of malignant cells (specifically leukemia cells) to nonmalignant macrophages (monocytes), and are useful in the treatment of leukemia. Additionally, Skowronski et al., 136 Endocrinology 20-26 (1995), have reported antiproliferative and differentiating actions of 1xcex1,25-dihydroxyvitamin D3 and other vitamin D3 analogs on prostate cancer cell lines.
Still other roles for vitamin D have been suggested in the modulation of the immune response (see, e.g., U.S. Pat. No. 4,749,710 issued to Truitt et al.; U.S. Pat. No. 5,559,107 issues to Gates et al., U.S. Pat. Nos. 5,540,919, 5,518,725 and 5,562,910 issued to Daynes et al.; U.S. Pat. No. 5,880,114 issued to DeLuca et al.) and the inflammatory response (see, e.g., U.S. Pat. No. 5,589,471 issued to Hansen et al.) as well as treatment of multiple sclerosis (see, U.S. Pat. No. 5,716,946 issued to DeLuca et al.).
Nonetheless, despite their activity in diverse biological functions the fact remains that at the levels required in vivo for effective use, e.g., as antileukemic agents, the known vitamin D compounds can induce markedly elevated and potentially dangerous blood calcium levels by virtue of their inherent calcemic activity. That is, the clinical use of active vitamin D compounds such as 1xcex1,25-dihydroxyvitamin D3 and other vitamin D3 analogs is precluded, or severely limited, because of their equally high potency as agents affecting calcium metabolism, i.e., by the risk of hypercalcemia. Considering the diverse biological actions of vitamin D and its potential as a therapeutic agent, a need exists for compounds with greater specific activity and selectivity of action, e.g., vitamin D compounds with antiproliferative and differentiating effects but which have less calcemic activity than therapeutic amounts of the known compounds or analogs of vitamin D.
The present invention provides a method for preparing hydroxy-25-ene-vitamin D compounds. These compounds are considered of value as pharmaceuticals because of their vitamin D activity but low toxicity when compared to the known vitamin D compounds. Specifically, these compounds are hydroxy-25-ene-vitamin D, such as 1xcex1-hydroxy-25-ene-vitamin D compounds and 24-hydroxy-25-ene-vitamin D compounds. These compounds are suitably prodrugs for 1xcex1,24-dihydroxylated vitamin D compounds as they are hydroxylated in vivo at the 24-position in the case of the 1xcex1-hydroxy-25-ene-vitamin D compounds and in the 1xcex1-position in the case of the 1xcex1-hydroxy-25-ene-vitamin D compounds to become the active forms of vitamin D. As prodrugs, these compounds in effect circumvent the first pass concern over intestinal vitamin D receptor binding which mediates intestinal calcium absorption, thereby resulting in reduced or no hypercalcemia compared with similar dosing with known active vitamin D compounds such as 1xcex1,25-dihydroxyvitamin D3.
The foregoing, and other advantages of the present invention, are realized in one aspect thereof in a method of making hydroxy-25-ene-vitamin D compounds. The 25-ene-vitamin D compounds are either 1xcex1-hydroxylated or 24-hydroxylated so that when administered to a human or an animal, they become dihydroxylated to active 1xcex1,24-dihydroxylated vitamin D compounds. The method includes reacting the appropriate vitamin D starting material with SO2 and protecting the hydroxyl functionality at C-3 and/or C-1 with t-butyldimethylsilyloxychloride to afford an SO2 adduct. Ozonolysis and reduction of the SO2 adduct severs the C-17 side chain and affords a truncated side chain C-22 alcohol. SO2 extrusion, and subsequent oxidation using the known Swern oxidation affords a C-22 aldehyde. The side chain is reassembled by reaction of the C-22 aldehyde with an appropriate phenyl sulfone to yield a 1xcex1-hydroxy-25-ene-vitamin D compound or a 25-ene-vitamin D compound depending upon the nature of the starting material.
If the 24-hydroxylated 25-ene-vitamin D compound is the desired end product, the 25-ene-vitamin D compound is incubated with human hepatoma cells, and the 24-hydroxy metabolite is isolated and purified to yield the 24(S)-25-ene-vitamin D compound.
Specifically, the invention provides a method of making hydroxy-25-ene-vitamin D compounds comprising the steps of reacting a 2,3-dimethyl-3-butene phenyl sulfone with a hydroxyl-protected C-22 aldehyde of a vitamin D, the vitamin D being hydroxyl-protected at C-3 or at C-3 and C-1.
The 2,3-dimethyl-3-butene phenyl sulfone is prepared by methylating, isomerizing and hydrolyzing ethyl dimethylacrylate to yield a dimethyl-3-ene-butanoic acid; amidating the dimethyl-3-ene-butanoic acid with oxazolidone to form oxazolidinones; separating the oxazolidinones to the desired isomer; oxidizing and reducing the desired isomer to yield a methyl-3-ene-butanol; reacting with methane sulfonyl chloride to form a mesylate; and substituting a phenyl sulfone group for the mesylate group to yield the 2,3-dimethyl-3-butene phenyl sulfone.
The hydroxyl-protected C-22 aldehyde of vitamin D is prepared by hydroxyl-protecting the C-3 position of vitamin D2 to yield a C-3 hydroxyl-protected vitamin D2; sulfonating the C-3 hydroxyl-protected vitamin D2 to yield a SO2 adduct; subjecting the adduct to SO2 extrusion to yield the trans-C-3-hydroxyl-protected vitamin D2; hydroxylating the trans-C-3-hydroxyl-protected vitamin D2 at the C-1 position; hydroxyl protecting the C-1 position; forming a SO2 adduct; truncating the C-17 sidechain to form a C-22 alcohol; and subjecting the C-22 alcohol to SO2 extrusion and Swern oxidation to form the C-22 aldehyde. The method of the present invention further includes reducing, isomerizing, deprotecting and irradiating the hydroyxl-protected-25-ene-vitamin D produced from the reaction of the phenyl sulfone and the C-22 aldehyde to yield a hydroxy-25-ene-vitamin D2.
If 25-ene-vitamin D2 compounds are desired, the hydroxyl-protected C-22 aldehyde of vitamin D is prepared by hydroxyl-protecting the C-3 position of vitamin D2 to yield a C-3 hydroxyl-protected vitamin D2; sulfonating the C-3 hydroxyl-protected vitamin D2 to yield a SO2 adduct; truncating the C-17 sidechain to form a C-22 alcohol; and subjecting the C-22 alcohol to SO2 extrusion and Swern oxidation to form the C-22 aldehyde.
The reaction of the C-22 aldehyde and the phenyl sulfone reaction yields a hydroxyl-protected-25-ene-vitamin D, which is reduced, isomerized and deprotected to yield a 25-ene-vitamin D2. If 24-hydroxy compounds are desired the 25-ene-vitamin D2 is further incubated with hepatoma cells to yield the 24-hydroxy-25-ene-vitamin D2.
It is noted that the starting material for the method of the present invention is suitably a vitamin D, a previtamin D, a cholesterol or an ergosterol.
Other advantages and a fuller appreciation of the specific attributes of this invention will be gained upon an examination of the following drawings, detailed description of preferred embodiments, and appended claims. It is expressly understood that the drawings are for the purpose of illustration and description only, and are not intended as a definition of the limits of the invention.